1. Field of the Invention
The present invention is generally directed to novel peptide-based compositions. More particularly, the present invention identifies new peptides that may be used to inhibit the binding of a key factor in the coagulation cascade, von Wilebrand factor (VWF) to platelets and to inhibit platelet adhesion and thrombus formation. As such, the present invention is directed to methods and compositions that may be used to inhibit thrombus formation and treat disorders associated with thrombus formation. Also, the new peptide-based compositions identified in the present invention may be used to inhibit the function of a family of intracellular proteins, named 14-3-3.
2. Background of the Related Art
Blood vessels operate under significant shear stresses that are a function of blood flow shear rate. Frequently, there is damage to small blood vessels and capillaries. When these vessels are damaged, hemostasis is triggered to stop the bleeding. Under typical circumstances, such an injury is dealt with through a sequence of events commonly referred to as the “thrombus formation”. Thrombus formation is dependent upon platelet adhesion, activation and aggregation and the coagulation cascade that culminates in the conversion of soluble fibrinogen to insoluble fibrin clot. Thrombus formation at site of wound prevents extravasation of blood components. Subsequently, wound healing and clot dissolution occurs and blood vessel integrity and flow is restored. Abnormal thrombus formation that causes obstruction of blood vessels is referred to as “thrombosis”.
A key step in the thrombus formation is platelet adhesion. In arteries and capillaries where blood flow shear rate is high, initial platelet adhesion is dependent on the binding of von Willebrand factor (VWF) to its platelet receptor, the glycoprotein (GP) Ib-IX-V complex (GPIb-IX) (Ruggeri, Prog Hemost Thromb 10, 35-68, 1991; Ware, Thromb Haemost 79,466-478, 1998). VWF binding to GPIb-IX mediates initial platelet adhesion to blood vessel wall, induces platelet activation and firm adhesion, leading platelet aggregation and formation of thrombus. Thus, inhibition of VWF binding to GPIb-IX leads to inhibition of thrombus formation.
GPIb-IX consists of four subunits, GPIbα, GPIbβ, GPIX and GPV. Extracellular domain of GPIba contains binding sites for VWF and thrombin. Binding of VWF to GPIb is regulated by cytoplasmic domain of GPIb-IX. A phosphoserine-dependent intracellular signaling molecule, ζ-form of 14-3-3 protein (Fu et al., Annu Rev Pharmacol Toxicol 40, 617-647, 2000), interacts with the cytoplasmic domain of GPIbα (Du et al., J Biol Chem 269, 18287-18290, 1994; Du et al., J Biol Chem 271, 7362-7367, 1996) and this interaction is dependent upon phosphorylation at Serine 609 of GPIbα (Bodnar et al., J. Biol. Chem. 274, 33474-33479, 1999). 14-3-3 is a family of intracellular signaling proteins that specifically recognize intracellular proteins that contains specific serine-phosphorylated 14-3-3 binding motifs. Different 14-3-3 binding proteins may have different sequences. However, these proteins are believed to bind to the same ligand binding pocket in 14-3-3. Thus, binding of one ligand may inhibit binding of a different ligand.
While thrombus formation is an essential mechanism by which unnecessary blood loss is avoided, this system often is dysregulated and leads to the formation of aberrant clots in the vasculature of a mammal. Thrombosis, is the physical condition that manifests when a thrombus is present in the vasculature of an animal. A thrombus (also called clot) is—gel-like or solidified blood formed by polymerized fibrin, platelets, and blood elements trapped by the fibrin-platelet net. While certain authorities imply a difference in the meaning of the terms “blood clot” and “thrombus,” these terms are typically employed interchangeably in the art to mean an aggregation as described above and the terms are used interchangeably herein.
The presence of thrombi in blood vessels can result in and/or from pathologies or treatments such as myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, percutaneous translumenal coronary angioplasty, athreosclerosis, disseminated intravascular coagulation, sepsis, pulmonary embolism and deep vein thrombosis. Blood clots also are seen on the surfaces of artificial organs, shunts and prostheses such as artificial heart valves that are implanted into an animal. In addition, certain pathological conditions (such as genetic lack of VWF cleaving protease, ADAMT13, causes spontaneous binding of VWF to platelets resulting in formation of microthrombi in blood vessels leading to thrombotic thrombocytopenic purpura and other microangiopathy. In order to combat the deleterious effects of thrombosis, anticoagulants, such as heparin are routinely administered. However, the problem with many existing anti-coagulants is that they fail to block platelet adhesion and aggregation, and can lead to uncontrolled bleeding or other complications. Therefore, there is a constant need to identify new and improved anti-thrombotic drugs.
The present invention is directed to new compositions that may be used as antithrombotics and/or anti-platelet agents.